1. Field of the Invention
The present invention relates to a method of quantitative determination of lipid. Further, the present invention relates to a simple method of quantitative determination of each of two components such as lipid and protein coexisting in a system without separating the components.
2. Related Background Art
Lipids are construction components of cells or organisms. Among the lipids, phospholipid is a principal lipid in construction of various membranes in the cells , such as plasma membrane, nuclear membrane, endoplasmic reticulum membrane, mitochondrial membrane, Golgi body membrane, lysosome membrane, and the like. The phospholipid is an amphiphilic molecule having both a polar group and a hydrophobic group in the molecule. When phospholipid is suspended in water, the polar groups become hydrated with water molecules, and the hydrophobic groups are excluded from the aqueous environment to cause gathering of the hydrophobic groups of the lipids. Consequently, micelles, lipid bilayers having a bimolecular membrane structure, or hexagonal II structures are formed: the state of gathering depending on the balance of the volumes of the hydrated hydrophilic groups and the hydrophobic groups. Among such structures, the lipid bilayer is the basic structure of biomembranes. The bilayer of phospholipid forms a closed vesicle (liposome), which is capable of incorporating membrane protein or the like as a constituent thereof and enclosing an aqueous phase therein. Therefore, the phospholipid bilayer structure is frequently employed as a model of a biomembrane for substance permeation, information transmission, and so forth. Further, the liposome is promising for use for a medicine capsule since the liposome is capable of retaining a water-soluble substance in its internal aqueous phase.
Many methods are known for quantitative determination of phospholipid, including a wet combustion method (Hoeflmayr, J. and Fried, R; Med. und Ern. 7, 9-10 (1966)) and a choline oxidase-phenol method (Takayama, M., Itoh, S., Nagasaki, T. and Tanimizu, I., Clin. Chim. Acta 79 93-98 (1977)). In the wet combustion method, the phospholipid is determined by adding sulfuric acid and a permanganate salt to a sample; heating the mixture in a boiling water bath to liberate the constituting phosphoric acid; adding ammonium molybdate and a reducing agent thereto; and measuring light absorbance caused by molybdenum blue formed thereby. In the choline oxidase-phenol method, the phospholipid is determined by reacting phospholipase D with the phospholipid in a sample to liberate the constituting choline; further reacting choline oxidase with the resulting choline to form betaine and hydrogen peroxide; and measuring light absorbance of the product of a quantitative condensation oxidation of phenol with 4-amino-antipyrine caused by hydrogen peroxide in the presence of peroxidase.
The above-mentioned wet combustion method has disadvantages of being troublesome in the operation of sequential addition of several reagents, and danger of bumping and measurement error by evaporation owing to heating in the presence of sulfuric acid in a boiling water bath. The choline oxidase-phenol method has disadvantages of instability of the enzyme, need for storing the reagents in a cold and dark condition, and insufficiency of reproducibility of measurement data obtained during lapse of time.
Generally, absorption spectrochemical quantitative analysis, in which the absorbance is measured at an absorption wavelength characteristic of the object of analysis in the sample, is widely used practically in quantitative determination of a substance because of simplicity of operation of direct measurement of absorbance, and needlessness of reacting the analysis object with another substance thus enabling the use of the sample later for another use.
However, this method of analysis has not been employed in the determination of lipids, because lipids such as phospholipid constituted of amphiphilic molecules form in an aqueous medium an aggregate in various state which scatters light and prevents precise assay of lipid concentration by light absorbance.
The constitution components of living matter such as lipids, proteins, nucleic acids, sugars are frequently handled in a coexisting state. Therefore, quantitative determination of such coexisting substances is an important operation.
Biomembranes have in a structure of a bilayered membrane are mainly constituted of lipid molecules, and protein gathers therein by non-covalent bonds, thereby functioning as a barrier for maintaining the interior medium. The ratio of lipid to protein in biomembranes differs greatly depending on the respective membranes. In mitochondrial membranes, for example, protein is contained 4 times as much as lipid. On the contrary, in some biomembranes, lipid is contained in an amount several times as much as protein. Accordingly, the ratio of lipid to protein in biomembrane in respective organs is an important object of study.
Various biomembrane models in which lipid and protein are coexisting are widely used in the study of the structure and the function of the biomembranes. The examples include proteoliposome which has closed vesicles constituted of artificial bimolecular lipid membrane containing therein protein; fine particles of glass beads or a high polymer which have lipid or protein adsorbed on the surface thereof; a planar lipid membrane formed in a small hole in a substrate made of TEFLON (tetrafluoroethylene fluoro carbon polymer) or the like (a black lipid membrane), or a planar bimolecular film reconstructed by sticking or patch-pipeting into which protein is incorporated; and Langmuir-Blodgett built-up films (LB built-up films) in which a lipid-protein coexisting system is built up in layers in a molecular level. In these artificial films also, the determination of the lipid and the protein is important in evaluating the function thereof.
If the optimum ratio of lipid to protein in natural, or artificially synthesized membrane structures can be obtained by quantitative determination, and a membrane can be synthesized effectively according to the derived ratio, then various membrane structure can be industrially synthesized at low cost without wasting valuable protein.
Further examples of lipid-protein coexisting systems include liposome, i.e., a closed vesicle composed of phospholipid membrane, in which a functional protein such as water-soluble enzyme is enclosed, and emulsions in which lipid and protein are simply mixed in a liquid medium.
Heretofore, in the determination of lipid and protein in a coexisting system, the determination of lipid is affected by the presence of protein, and the determination of protein is affected by the presence of lipid. Therefore, lipid and protein could not be precisely determined in a coexisting state. In determination of lipid by the above-mentioned absorbance measurement, the influence of the absorbance of coexisting protein is not negligible.
Various methods for determining protein are known, including colorimetry utilizing a metal ion, UV absorption method based on tyrosine and triptophan, and UV absorption method based on peptide bonds. A typical method of the colorimetry is a Lowery method (J. Biol. Chim., vol. 193, p265 (1951)) employing a phenol reagent. This method utilizes a blue color developed by reaction of a phenol with protein. The main component of the reagent is phosphomolybdate or phosphotungstate, a complicated complex formed from the metal oxide and phosphoric acid. This complex reacts with a reducing agent such as protein to develop blue color of phosphomolybdate blue or phosphotungstate blue. The protein is determined by measurement of the absorbance caused by the color. In a color reaction method called a BCA method, protein present converts a cupric ion into a cuprous ion in an alkaline medium, and the cuprous ion forms a purple complex compound with bicinchoninic acid molecule (i.e., BCA: 4,4'-dicarboxy-2,2'-biquinoline). The protein is determined according to the absorbance of this complex compound.
On the contrary, the UV absorption method for determining protein includes measurement of absorption at 280 nm caused by tyrosine or triptophan, and measurement of absorption at 215 to 225 nm caused by the peptide linkage. Both measurement methods are advantageous in that the operation is simple and the sample is not lost in comparison with color development methods, but are disadvantageous in that the measurement of protein is based on the content of tyrosine or triptophan and the determination result varies greatly depending on the kinds of the protein and is susceptible to an interfering substance such as nucleic acid which has absorption in this absorption region.
In these known methods of determining protein, coexisting lipid also undergoes reaction to develop color. For example, in the Lowry method and other methods employing a phenol reagent, the phosphomolybdate is reduced by the protein to develop a blue color of phosphomolybdate blue, and simultaneously the coexisting lipid which exhibits reduction activity like the protein develops color similarly.
Accordingly, in the quantitative determination of each of lipid and protein coexisting in a system, they have to be separated before the determination of lipid and protein.
The separation is usually conducted by an extraction method in which the lipid component in the sample is dissolved in an organic solvent layer and is recovered as an organic solvent fraction, and the protein component insoluble in the organic solvent is recovered as an aqueous fraction. The obtained organic solvent fraction is concentrated and evaporated to dryness, and is quantitatively determined as the lipid fraction. The aqueous fraction containing the protein is concentrated and the protein therein is quantitatively determined by various methods.
Thus the conventional determination of lipid and protein coexisting in a system is extremely complicated.
In conventional methods, in spite of the complicated operations, the protein is liable to enter the organic solvent layer, and the lipid is liable to enter the aqueous layer in some proportions since the extraction with an organic solvent is based on equilibrated partition, which causes a determination error frequently. Moreover, loss of the sample is not negligible in the extraction process, so that a relatively large amount of a sample is required. Furthermore, in determination of lipid and protein contained in a membrane structure, strongly hydrophobic protein such as membrane protein may contaminate the organic solvent fraction in high probability, and the lipid and the protein may possibly exist in an unseparated state in the organic layer, which prevents precise quantitative determination.
The above discussion relates to a two component system containing lipid and protein. The same discussion may be made on a two-component system containing two components selected from lipids, sugars, nucleic acids, pigments and various medicines.